Solubilization of  msw with blend enzymes

ABSTRACT

The present invention relates to a method for solubilisation or hydrolysis of Municipal Solid Waste (MSW) with an enzyme blend and an enzyme composition for solubilization of Municipal Solid Waste (MSW), the enzyme composition comprising a cellulolytic background composition and a protease, lipase and/or beta-glucanase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from European Patent Application No.14182698.2 filed Aug. 28, 2014. The content of this application is fullyincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method for solubilisation orhydrolysis of Municipal Solid Waste (MSW) with an enzyme blend,optionally for subsequent production of biogas and/or bioethanol.

BACKGROUND OF THE INVENTION

Municipal Solid Waste (MSW) is commonly also known as trash, garbage,refuse or rubbish. It consists of solid waste fractions that typicallycomes from municipalities and includes for instance waste from homes,schools, offices, hospitals, institutions etc. MSW is producedworld-wide in very large quantities; thus in EU alone 2.44 million tonswere generated in 2012 (Eurostat, 2014). The challenges of MSWproduction are many and may include collection, sorting, treatment, anddisposal. Furthermore, well known environmental issues such as air andgroundwater pollution from landfills is related to MSW. With anincreasing world population entailing an increasing waste production,proper sustainable MSW management is a global challenge.

Although being environmental troublesome MSW also represents a largeunexploited resource that may be used for energy production andrecycling/recovery of scarce resources. Incineration is a technologythat is widely used in some European countries, for instance Denmark,Sweden and Germany. The generation of energy is highly efficient butrecovery of materials is limited. Furthermore, incineration of MSWresults in a large production of slag (ash) which for some types ofwaste fractions can have environmental negative impacts (Idris and Saed2002, Journal of Hazardous Materials B93 201-208). Capture of gasgenerated from anaerobic digestion of organic material at landfills isanother way of generating energy from MSW but this technology also has avery low efficiency in materials recycling.

Integrated processes with concurrent energy production and recycling ofmaterials are an attractive solution that has become a lot of attentionlately. In such waste refineries, organic parts of MSW represent aresource that potentially can be used for bioenergy production in theform of for instance biogas (Hartmann and Ahring, 2006, Water Science &Technology Vol 53 No 8 p. 7-22). However, sorting of MSW in organicfractions for bioenergy production and plastic/metal fractions formaterial recovery is not easy due to the very inhomogeneous nature ofMSW. As described by Jensen et al. (2012) (WO 2013/185777) presorting ofMSW is typically costly, inefficient or impractical while source-sortingrequires large infrastructure and operating expenses as well as activeparticipation from the community from which the waste is collected.

Enzymatic treatment of MSW has lately been described and seems like avery interesting and innovative approach in the MSW management (Jensenet al. 2010, Waste Management 30, p. 2497-2503; Jensen et al. 2011,Biochem Biotechnol 165, p. 1799-1811; Tonini and Astrup 2012, WasteManagement 32, p. 165-176). This technology is based on aliquefaction/solubilization step of organic degradable parts withhydrolytic enzymes and a subsequent separation of the MSW into abioliquid and solids. The bioliquid can be used for biogas productionwhile the solids can be further sorted and used for recycling orcombusted according to the composition of the material. The technologyhas proven very robust at even high dry matter concentrations (35%) andhas been demonstrated in pilot/demonstration facilities treating up to 1ton of MSW/hour.

Tonini and Astrup (2012), evaluated the environmentalsustainability—using life-cycle assessment—of four different wasterefinery scenarios using this enzymatic liquefaction technology. Theirassessment was based on a pilot-scale facility established at a Danishincinerator Amagerforbrænding in Copenhagen, Denmark. The differentscenarios were compared to incineration. The authors concluded that“enzymatic refining of the waste with utilization of the products forenergy recovery can represent a valuable alternative to incinerationfrom both an energy and environmental point of view. This is the case ifthe downstream energy options for exploiting the solid and liquidfractions are co-combustion and anaerobic digestion for biogasproduction”. The authors also concluded that cost savings of the wasterefinery was related to a higher recovery of metals and energy.Furthermore, “improvement in the environmental as well as energyperformance of the waste refinery itself was primarily related to theoptimization of energy and enzymes consumption”.

The use of cellulases (for instance Novozymes A/S Celluclast® 1.5 L,Novozymes A/S CELLIC® Ctec2 and Novozymes A/S CELLIC® Ctec3) forliquefaction of MSW with subsequent separation of unsorted waste into abioliquid—used for biogas production—and into inorganic valuableproducts suitable for recycling has been clearly illustrated(WO2013/185777A1, the content of which is hereby incorporated byreference). However, with MSW being a complex substrate containing otherorganic components than cellulose (e.g. protein and lipids) it seemsreasonable that the liquefaction process can be further improved bysupplementing cellulases with other enzyme activities. Until now thistheory has not been proven. An attempt was made Jensen et al. 2010 whotested a protease (Novozymes A/S Alcalase 2.5 L) and a α-amylase(Novozymes A/S Liquozyme® SC DC) as single enzymes and in combinationwith a cellulase (Novozymes A/S Celluclast® 1.5 L). “The cellulyticenzyme was the key catalyst in the liquefaction of degradable fractionsboth in terms of lowering viscosity and particle size distribution”. Noeffect of the α-amylase and protease and interaction in between and withthe cellulase was found. Nevertheless, it would be beneficial to providean enzymes mix with a higher efficiency for liquefying MSW than thecellulases previously tested (Celluclast® 1.5L, CELLIC® Ctec2 andCELLIC® Ctec3). Such invention could contribute to a global change inMSW management practices and turn an environmental problem into aprofitable and environmentally beneficial solution.

SUMMARY OF THE INVENTION

The present invention relates to an enzyme composition forsolubilization of Municipal Solid Waste (MSW), the enzyme compositioncomprising a cellulolytic background composition (CBC), and one or moreenzymes selected from (i) a protease; (ii) a lipase and (iii) abeta-glucanase. In one embodiment, the composition further comprises oneor more enzymes selected from (iv) a pectate lyase; (v) a mannanase and(vi) an amylase.

In one embodiment of the invention, the cellulolytic backgroundcomposition (CBC) comprises one or more enzymes selected from a) acellobiohydrolase I or variant thereof; (b) cellobiohydrolase II orvariant thereof; (c) beta-glucosidase or variant thereof; and (d) apolypeptide having cellulolytic enhancing activity; or homologs thereof.In a further embodiment of the invention the cellulolytic backgroundcomposition comprise one or more enzymes selected from (a) anAspergillus fumigatus cellobiohydrolase I or variant thereof; (b) anAspergillus fumigatus cellobiohydrolase II or variant thereof; (c) anAspergillus fumigatus beta-glucosidase or variant thereof; and (d) aPenicillium sp. GH61 polypeptide having cellulolytic enhancing activity;or homologs thereof.

In a related embodiment of the invention the (i) a protease is derivedfrom the genus Bacillus, such as e.g. Bacillus amyloliquefaciens such ase.g. the protease encoded by SEQ ID NO: 1, or a protease having at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 1.

In a related embodiment of the invention the (ii) a lipase is derivedfrom the genus Thermomyces sp. such as e.g. Thermomyces lanuginosus suchas e.g. the lipase encoded by SEQ ID NO: 2 (or a lipase having at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to SEQ ID NO: 2) or wherein the(ii) a lipase is derived from the genus Humicola sp. such as e.g.Humicola insolens (or a lipase having at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the Humicola insolens lipase). In a related embodiment ofthe invention the (iii) a beta-glucanase is derived from a member of thegenus Aspergillus such as e.g. Aspergillus aculeatus such as e.g. thebeta-glucanase encoded by the sequence encoded by SEQ ID NO: 4 orhomologs thereof (e.g., a beta-glucanase having at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 4). In a related embodiment of theinvention the (iv) a pectate lyase forms part of a multicomponent enzymecomposition comprising pectate lyase, xylanase and cellulase activitiessuch as e.g. Novozym 81243™. In a related embodiment of the inventionthe (v) a mannanase is an endo-mannosidase derived from the genusBacillus such as e.g. Bacillus bogoriensis such as e.g. theendo-mannosidase encoded by SEQ ID NO: 6 or homologs thereof (e.g., anendo-mannosidase having at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 6). In a related embodiment of the invention the (vi) an amylaseis an alpha-amylase derived from the genus Rhozimucor such as e.g.Rhizomucor pusillus such as e.g. the alpha-amylase encoded by SEQ ID NO:5 or homologs thereof (e.g., an alpha-amylase having at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 5).

In yet a related embodiment, the protease is present at a ratio between0-20% w/w, such as e.g. 10% w/w of the total enzyme protein. In arelated embodiment of the invention the the beta-glucanase is present ata ratio between 0-30% w/w, such as e.g. 15% w/w of the total enzymeprotein. In a yet another related embodiment of the invention thepectate-lyase is present at a ratio between 0-10% w/w, such as e.g. 5%w/w of the total enzyme protein. In a yet another related embodiment ofthe invention the mannanase or amylase is present at a ratio between0-10% w/w, such as e.g. 5% w/w of the total enzyme protein. In a yetanother related embodiment of the invention the cellulolytic enzymeblend is present at a ratio between 40%-99% w/w, such as e.g. between50%-90% w/w, such as e.g. 60%-80% w/w, such as e.g. 65-75% of the totalenzyme protein. In a yet another related embodiment of the invention theenzyme composition further comprises one or more enzymes selected from acellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducibleprotein (CIP) an esterase, an expansin, a ligninolytic enzyme, anoxidoreductase, a pectinase, a protease, and a swollenin. In a yetanother related embodiment of the invention the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

A related aspect of present invention relates to a process forsolubilizing waste comprising: contacting waste with the enzymecomposition of the invention, wherein the waste may be Municipal SolidWaste (MSW).

Yet a related aspect of the invention relates to a process for producinga fermentation product, comprising: (a) treating MSW with the enzymecomposition of the present invention, (b) fermenting the solubilizedand/or hydrolysed MSW with one or more fermenting microorganisms toproduce a fermentation product; and, (c) recovering the fermentationproduct from the fermentation. The waste in said process may bepretreated.

We have tested a variety of commercial enzymes on a model-substrate ofMSW and used dry matter solubilisation of the substrate as a parameterfor the liquefaction effect of the enzymes. The model waste simulatedthe organic fractions of MSW (based on a publication of the compositionof Danish MSW; Riber et al. 2009, Waste Management 29, p. 1251-1257),and consisted of a vegetable fraction (eg. Carrots, potatoes, cerealsetc.) animal by-product fraction (e.g. cheese and meats) and cellulosefraction (e.g. paper, card-board, textile).

The screening experiments were carried out in 20 gram scale at 50° C.for 24 hours. Some specific enzymes improved the dry mattersolubilisation of the model waste when they replaced parts of theCellulolytic Background Composition (CBC), including some proteases,lipases and beta-glucanases. Subsequently, candidates were selected forfurther testing in blending experiments.

The invention provides a process for solubilizing MSW by adding one ormore enzymes—including acid protease, acid lipase and acidbeta-glucanase—in combination with a cellulase composition at a suitabletemperature and pH to MSW.

As apparent from the findings disclosed herein, it was surprisinglyfound that a synergistic effect in solubilization of the MSW wasobtained when adding blends of different enzymes to a cellulolyticbackground composition (27.1%), compared to the individual contributionsof e.g. components B.a protease, T.l pholip and A.a BG which was up to5%, 8.5% and 8.2% respectively.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows solubilization of model waste in free fall experiments attwo different dry matter concentrations. The figure shows thedistribution of dry matter liquid (grey bars) and solids fractions(white bars).

FIG. 2 shows a plot of data from dose response experiments with blendenzymes and CBC and model waste. The figure illustrates the dry matterfound in the liquid fraction (TS solubilization) at different enzymesconcentration.

FIG. 3 shows a plot of data from dose response experiments with blendenzymes and CBC and model waste. The figure illustrates sum of glucoseand xylose (g/l) at different enzymes concentration.

FIG. 4 shows a graph illustrating the effect of removing either B.aprotease, T.l pholip or A.a BG components from the optimized blend. Thefigure illustrates the dry matter found in the liquid fraction (TSsolubilization).

FIG. 5 shows a data table from dose response experiments with blendenzymes and CBC and model waste. The table illustrates the amount ofglucose, xylose and lactic acid at different enzyme concentrations.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. Acetylxylan esteraseactivity can be determined using 0.5 mM p-nitrophenylacetate assubstrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN™ 20(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esteraseis defined as the amount of enzyme capable of releasing 1 μmole ofp-nitrophenolate anion per minute at pH 5, 25° C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase.Alpha-L-arabinofuranosidase activity can be determined using 5 mg ofmedium viscosity wheat arabinoxylan (Megazyme International Ireland,Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5in a total volume of 200 μl for 30 minutes at 40° C. followed byarabinose analysis by AMINEX® HPX-87H column chromatography (Bio-RadLaboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. Alpha-glucuronidase activity can be determined according to deVries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidaseequals the amount of enzyme capable of releasing 1 μmole of glucuronicor 4-O-methylglucuronic acid per minute at pH 5, 40° C.

Auxiliary Activity 9 polypeptide: The term “Auxiliary Activity 9polypeptide” or “AA9 polypeptide” means a polypeptide classified as alytic polysaccharide monooxygenase (Quinlan et al., 2011, Proc. Natl.Acad. Sci. USA 208: 15079-15084; Phillips et al., 2011, ACS Chem. Biol.6: 1399-1406; Lin et al., 2012, Structure 20: 1051-1061). AA9polypeptides were formerly classified into the glycoside hydrolaseFamily 61 (GH61) according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

AA9 polypeptides enhance the hydrolysis of a cellulosic material by anenzyme having cellulolytic activity. Cellulolytic enhancing activity canbe determined by measuring the increase in reducing sugars or theincrease of the total of cellobiose and glucose from the hydrolysis of acellulosic material by cellulolytic enzyme under the followingconditions: 1-50 mg of total protein/g of cellulose in pretreated cornstover (PCS), wherein total protein is comprised of 50-99.5% w/wcellulolytic enzyme protein and 0.5-50% w/w protein of an AA9polypeptide for 1-7 days at a suitable temperature, such as 40° C.-80°C., e.g., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75°C., or 80° C. and a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis withequal total protein loading without cellulolytic enhancing activity(1-50 mg of cellulolytic protein/g of cellulose in PCS).

AA9 polypeptide enhancing activity can be determined using a mixture ofCELLUCLAST® 1.5L (Novozymes A/S, Bagsværd, Denmark) and beta-glucosidaseas the source of the cellulolytic activity, wherein the beta-glucosidaseis present at a weight of at least 2-5% protein of the cellulase proteinloading. In one aspect, the beta-glucosidase is an Aspergillus oryzaebeta-glucosidase (e.g., recombinantly produced in Aspergillus oryzaeaccording to WO 02/095014). In another aspect, the beta-glucosidase isan Aspergillus fumigatus beta-glucosidase (e.g., recombinantly producedin Aspergillus oryzae as described in WO 02/095014).

AA9 polypeptide enhancing activity can also be determined by incubatingan AA9 polypeptide with 0.5% phosphoric acid swollen cellulose (PASC),100 mM sodium acetate pH 5, 1 mM MnSO₄, 0.1% gallic acid, 0.025 mg/ml ofAspergillus fumigatus beta-glucosidase, and 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hoursat 40° C. followed by determination of the glucose released from thePASC.

AA9 polypeptide enhancing activity can also be determined according toWO 2013/028928 for high temperature compositions.

AA9 polypeptides enhance the hydrolysis of a cellulosic materialcatalyzed by enzyme having cellulolytic activity by reducing the amountof cellulolytic enzyme required to reach the same degree of hydrolysispreferably at least 1.01-fold, e.g., at least 1.05-fold, at least1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or atleast 20-fold.

The AA9 polypeptide can also be used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043 or WO2012/122518, e.g., manganese or copper.

The AA9 polypeptide can be used in the presence of a dioxy compound, abicylic compound, a heterocyclic compound, a nitrogen-containingcompound, a quinone compound, a sulfur-containing compound, or a liquorobtained from a pretreated cellulosic or hemicellulosic material such aspretreated corn stover (WO 2012/021394, WO 2012/021395, WO 2012/021396,WO 2012/021399, WO 2012/021400, WO 2012/021401, WO 2012/021408, and WO2012/021410).

Beta-glucanase: The term “beta-glucanase” means any type ofendo-beta-glucanase that hydrolyzes (1,3)- or (1,4)-linkages inbeta-D-glucans (E.C. 3.2.1.73) (E.C.3.2.1.6).

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.Beta-glucosidase activity can be determined usingp-nitrophenyl-beta-D-glucopyranoside as substrate according to theprocedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. Oneunit of beta-glucosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. Beta-xylosidase activity can be determinedusing 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodiumcitrate containing 0.01% TWEEN® 20 at pH 5, 40° C. One unit ofbeta-xylosidase is defined as 1.0 μmole of p-nitrophenolate anionproduced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside in 100 mM sodium citrate containing 0.01%TWEEN® 20.

Binding domain: The term “binding domain” e.g., “cellulose bindingdomain” means the region of an enzyme that mediates binding of theenzyme to amorphous regions of a cellulose substrate. The cellulosebinding domain (CBD) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

Carbohydrate binding module: The term “carbohydrate binding module”means a domain within a carbohydrate-active enzyme that providescarbohydrate-binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). A majority of known carbohydrate binding modules (CBMs) arecontiguous amino acid sequences with a discrete fold. The carbohydratebinding module (CBM) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme. Some CBMs are known to havespecificity for cellulose.

Catalase: The term “catalase” means ahydrogen-peroxide:hydrogen-peroxide oxidoreductase (EC 1.11.1.6) thatcatalyzes the conversion of 2 H₂O₂ to O₂+2 H₂O. For purposes of thepresent invention, catalase activity is determined according to U.S.Pat. No. 5,646,025. One unit of catalase activity equals the amount ofenzyme that catalyzes the oxidation of 1 μmole of hydrogen peroxideunder the assay conditions.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity canbe determined according to the procedures described by Lever et al.,1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

Cellulolytic Background Composition (CBC) or Cellulolytic Enzyme Blend:

The term “Cellulolytic background composition” or “CBC” means an enzymecomposition comprising a mixture of two or more cellulolytic enzymes. Inone embodiment the CBC comprises two or more cellulolytic enzymesselected from: i) an Aspergillus fumigatus cellobiohydrolase I; (ii) anAspergillus fumigatus cellobiohydrolase II; (iii) an Aspergillusfumigatus beta-glucosidase or variant thereof; and (iv) a Penicilliumsp. GH61 polypeptide having cellulolytic enhancing activity; or homologsthereof. The CBC may further comprise one or more enzymes selected from:(a) an Aspergillus fumigatus xylanase or homolog thereof, (b) anAspergillus fumigatus beta-xylosidase or homolog thereof; or (c) acombination of (a) and (b) (as described in further detail in WO2013/028928). The CBC may be any CBC described in WO 2013/028928 (thecontent of which is hereby incorporated by reference). In oneembodiment, the CBC is CELLIC® Ctec3 obtainable from Novozymes A/S(Bagsværd, Denmark).

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzymeactivity can be measured using insoluble substrates, including WhatmanN°1 filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman N°1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (chose, 1987,Pure Appl. Chem. 59: 257-68).

Cellulolytic enzyme activity can be determined by measuring the increasein production/release of sugars during hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in pretreated cornstover (PCS) (or other pretreated cellulosic material) for 3-7 days at asuitable temperature such as 40° C.-80° C., e.g., 40° C., 45° C., 50°C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C., and a suitablepH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,or 9.0, compared to a control hydrolysis without addition ofcellulolytic enzyme protein. Typical conditions are 1 ml reactions,washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodiumacetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugaranalysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories,Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose.

In an embodiment, the cellulosic material is agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, or wood (including forestryresidue).

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Dissolved Oxygen Saturation Level: The saturation level of oxygen isdetermined at the standard partial pressure (0.21 atmosphere) of oxygen.The saturation level at the standard partial pressure of oxygen isdependent on the temperature and solute concentrations. In an embodimentwhere the temperature during hydrolysis is 50° C., the saturation levelwould typically be in the range of 5-5.5 mg oxygen per kg slurry,depending on the solute concentrations. Hence, a concentration ofdissolved oxygen of 0.5 to 10% of the saturation level at 50° C.corresponds to an amount of dissolved oxygen in a range from 0.025 ppm(0.5×5/100) to 0.55 ppm (10×5.5/100), such as, e.g., 0.05 to 0.165 ppm,and a concentration of dissolved oxygen of 10-70% of the saturationlevel at 50° C. corresponds to an amount of dissolved oxygen in a rangefrom 0.50 ppm (10×5/100) to 3.85 ppm (70×5.5/100), such as, e.g., 1 to 2ppm. In an embodiment, oxygen is added in an amount in the range of 0.5to 5 ppm, such as 0.5 to 4.5 ppm, 0.5 to 4 ppm, 0.5 to 3.5 ppm, 0.5 to 3ppm, 0.5 to 2.5 ppm, or 0.5 to 2 ppm.

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). Endoglucanase activity can also bedetermined using carboxymethyl cellulose (CMC) as substrate according tothe procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5,40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase (FAE) is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. Feruloyl esterase activity can be determinedusing 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetatepH 5.0. One unit of feruloyl esterase equals the amount of enzymecapable of releasing 1 μmole of p-nitrophenolate anion per minute at pH5, 25° C.

Fragment: The term “fragment” means a polypeptide or a catalytic orbinding domain having one or more (e.g., several) amino acids absentfrom the amino and/or carboxyl terminus of a mature polypeptide ordomain; wherein the fragment has enzymatic or substrate bindingactivity.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Current Opinion In Microbiology 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature such as 40° C.-80° C., e.g., 40°C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C.,and a suitable pH such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, or 9.0.

Hemicellulosic material: The term “hemicellulosic material” means anymaterial comprising hemicelluloses.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Lipase: The term “lipase” means any enzyme that catalyzes the hydrolysisof lipids and/or having hydrolytic activity in class EC 3.1.1.—asdefined by Enzyme Nomenclature. Particular useful is triacyl glycerollipases (E.C.3.1.1.3) and phospholipase A1 (EC 3.1.1.32) andphospholipase A2 (E.C.3.1.1.4), but also other phospholipases(E.C.3.1.1.5), (E.C.3.1.4.4), (E.C.3.1.4.11), (E.C.3.1.4.50),(E.C.3.1.4.54).

Mannanases: In the context of the present invention a “mannanase” is abeta-mannanase and defined as an enzyme belonging to EC 3.2.1.78 orE.C.3.2.1.25. Mannanases have been identified in several Bacillusorganisms. For example, Talbot et al., Appl. Environ. Microbiol., Vol.56, No. 11, pp. 3505-3510 (1990) describes a beta-mannanase derived fromBacillus stearothermophilus having an optimum pH of 5.5-7.5. Mendoza etal., World J. Microbiol. Biotech., Vol. 10, No. 5, pp. 551-555 (1994)describes a beta-mannanase derived from Bacillus subtilis having anoptimum activity at pH 5.0 and 55° C. JP-03047076 discloses abeta-mannanase derived from Bacillus sp., having an optimum pH of 8-10.JP-63056289 describes the production of an alkaline, thermostablebeta-mannanase. JP-08051975 discloses alkaline beta-mannanases fromalkalophilic Bacillus sp. AM-001. A purified mannanase from Bacillusamyloliquefaciens is disclosed in WO 97/11164. WO 94/25576 discloses anenzyme from Aspergillus aculeatus, CBS 101.43, exhibiting mannanaseactivity and WO 93/24622 discloses a mannanase isolated from Trichodermareesei.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving enzymatic activity.

Municipal Solid Waste (MSW): The term “municipal solid waste” or “MSW”is intended to mean solid waste fractions that is typically available inmunicipalities (cities, towns, villages). MSW can be a combination ofplant materials (fruit, vegetables, grains, corn etc), animal materials(meats etc.), cellulosic material (paper, cardboard, diapers, textileetc.), glass, plastic, metal. MSW includes the following but is notlimited to any one or more of the following:

waste collected from homes, schools, hospitals, offices, business,industries such as restaurants and food processing industries. MSW canpotentially have been treated by shredding or pulping devices.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Pretreated municipal solid waste material: The term “pretreatedmunicipal solid waste material” means a municipal solid waste materialderived from biomass by treatment with heat and dilute sulfuric acid,alkaline pretreatment, neutral pretreatment, or any pretreatment knownin the art.

Protease: The term “protease” means any protease or proteolytic enzymesuitable for use under neutral or acidic conditions. Suitable proteasesinclude those of animal, vegetable or microbial origin. Chemically orgenetically modified mutants are included. Suitable proteases includesmetallo endoprotease that hydrolyzes internal peptide bonds (E.C.3.4.24.28), serine endoprotease that hydrolyzes internal peptide bonds(E.C:3.4.23.23), endoprotease that hydrolyzes peptide bonds at thecarboxy side of lysine and arginine residues E.C.3.4.21.4),aminopeptidase (E.C. 3.4.11.1) and exopeptidase that liberates aminoacids by hydrolysis of the N-terminal peptide bond (E.C. 3.4.11.1).

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Solubilization: The term “solubilization” means enzymatic treatment of asubstrate. In present disclosure, the terms “hydrolyzation”,“liquefaction”, “saccharification” and “solubilization” may be usedinterchangeably.

Variant: The term “variant” means a polypeptide comprising analteration, i.e., a substitution, insertion, and/or deletion, at one ormore (e.g., several) positions. A substitution means replacement of theamino acid occupying a position with a different amino acid; a deletionmeans removal of the amino acid occupying a position; and an insertionmeans adding an amino acid adjacent to and immediately following theamino acid occupying a position.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrimann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. A common total xylanolytic activity assay is based onproduction of reducing sugars from polymeric 4-O-methyl glucuronoxylanas described in Bailey et al., 1992, Interlaboratory testing of methodsfor assay of xylanase activity, Journal of Biotechnology 23(3): 257-270.Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan assubstrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μmole of azurineproduced per minute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan assubstrate in 200 mM sodium phosphate pH 6.

Xylan degrading activity can be determined by measuring the increase inhydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo.,USA) by xylan-degrading enzyme(s) under the following typicalconditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg ofxylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50° C.,24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH)assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. Xylanase activity can be determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Reference to “about” a value or parameter herein includes aspects thatare directed to that value or parameter per se. For example, descriptionreferring to “about X” includes the aspect “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that the aspects of the invention describedherein include “consisting” and/or “consisting essentially of” aspects.

Unless defined otherwise or clearly indicated by context, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments of the invention enzymatic solubilization of MSW iscarried out together with natural occurring microorganisms found in thewaste (concurrent enzymatic and microbial hydrolysis and fermentation)or found in recycled process wastes/solutions.

In some embodiments the microbial growth has a pH lowering effectespecially when metabolites like caboxylic acids and fatty acids (e.g.acetate, propionate, butyrate, lactate) is produced.

In other embodiments of the invention it might be an advantage toinoculate MSW using different microbial species. These might includemicroorganisms that shows extra-cellular cellulase activities,microorganisms capable of degrading lignin, acetate-producingmicroorganisms, propionate-producing microorganisms, butyrate-producingorganisms, ethanol-producing microorganisms and lactate producingmicroorganisms. Such embodiments are further described in DONG patentpage 21-25 (WO2013/185777, the content of which is hereby incorporatedby reference)

In practicing embodiments of the invention it can be advantageous toadjust temperature and water and dry matter content of the MSW. Enzymesnormally show an optimal temperature and dry matter range. Hydrolysis ofMSW is normally performed with agitation. This can be in reactorsproviding agitation by free fall mixing (as also described by DONGWO2006/056838 and WO2011/032557), stirred-tank reactors or similarsystems. Suitable process time, temperature and pH conditions canreadily be determined by one skilled in the art and is dependent on MSWcomposition, dry matter concentration and enzyme.

The present invention is also directed to processes for using thecompositions thereof.

The present invention also relates to processes for degrading amunicipal solid waste material, comprising: treating the municipal solidwaste material with an enzyme composition comprising a cellulolyticbackground composition combined with one or more enzymes selected from(i) a protease; (ii) a lipase and (iii) a beta-glucanase; and optionallycombined with one or more further enzymes selected from (iv) a pectatelyase; (v) a mannanase and (vi) an amylase. In one aspect, the processesfurther comprise recovering the degraded municipal solid waste material.Soluble products of degradation of the municipal solid waste materialcan be separated from insoluble municipal solid waste material using amethod known in the art such as, for example, centrifugation,filtration, or gravity settling. In preferred embodiments solubilizedcompounds can be converted to Biogas (mainly comprising CH₄ and CO₂) byanaerobic digestion. In other embodiments solubilized sugars can beconverted to ethanol by fermentation.

The present invention also relates to processes of producing afermentation product, comprising: (a) solubilizing a municipal solidwaste material with an enzyme composition comprising a cellulolyticbackground composition combined with one or more enzymes selected from(i) a protease; (ii) a lipase and (iii) a beta-glucanase; and optionallycombined with one or more further enzymes selected from (iv) a pectatelyase; (v) a mannanase and (vi) an amylase; (b) fermenting thesolubilized municipal solid waste material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation. Inpreferred embodiments solubilized compounds can be converted to biogas(mainly comprising CH₄ and CO₂) by anaerobic digestion. In otherembodiments solubilized sugars can be converted to ethanol byfermentation.

The present invention also relates to processes of fermenting amunicipal solid waste material, comprising: fermenting the municipalsolid waste material with one or more (e.g., several) fermentingmicroorganisms, wherein the municipal solid waste material issaccharified with an enzyme composition comprising a cellulolyticbackground composition combined with one or more enzymes selected from(i) a protease; (ii) a lipase and (iii) a beta-glucanase; and optionallycombined with one or more further enzymes selected from (iv) a pectatelyase; (v) a mannanase and (vi) an amylase. In one aspect, thefermenting of the municipal solid waste material produces a fermentationproduct. In another aspect, the processes further comprise recoveringthe fermentation product from the fermentation. In preferred embodimentssolubilized compounds can be converted to Biogas (mainly comprising CH₄and CO₂) by anaerobic digestion. In other embodiments solubilized sugarscan be converted to ethanol by fermentation.

The processes of the present invention can also be used to solubilizethe municipal solid waste material to fermentable sugars and to convertthe fermentable sugars to many useful fermentation products, e.g., fuel(ethanol, n-butanol, isobutanol, biodiesel, jet fuel) and/or platformchemicals (e.g., acids, alcohols, ketones, gases, oils, and the like).The production of a desired fermentation product from the municipalsolid waste material typically involves enzymatic solubilization andfermentation.

The processing of the municipal solid waste material according to thepresent invention can be accomplished using methods conventional in theart. Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Solubilization and fermentation, separate or simultaneous, include, butare not limited to, separate hydrolysis and fermentation (SHF);simultaneous saccharification and fermentation (SSF); simultaneoussaccharification and co-fermentation (SSCF); hybrid hydrolysis andfermentation (HHF); separate hydrolysis and co-fermentation (SHCF);hybrid hydrolysis and co-fermentation (HHCF); and direct microbialconversion (DMC), also sometimes called consolidated bioprocessing(CBP). SHF uses separate process steps to first enzymatically hydrolyzethe municipal solid waste material to fermentable sugars, e.g., glucose,cellobiose, and pentose monomers, and then ferment the fermentablesugars to ethanol. In SSF, the enzymatic hydrolysis of the municipalsolid waste material and the fermentation of sugars to ethanol arecombined in one step (Philippidis, G. P., 1996, Cellulose bioconversiontechnology, in Handbook on Bioethanol: Production and Utilization,Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212). SSCFinvolves the co-fermentation of multiple sugars (Sheehan and Himmel,1999, Biotechnol. Prog. 15: 817-827). HHF involves a separate hydrolysisstep, and in addition a simultaneous saccharification and hydrolysisstep, which can be carried out in the same reactor. The steps in an HHFprocess can be carried out at different temperatures, i.e., hightemperature enzymatic saccharification followed by SSF at a lowertemperature that the fermentation strain can tolerate. DMC combines allthree processes (enzyme production, hydrolysis, and fermentation) in oneor more (e.g., several) steps where the same organism is used to producethe enzymes for conversion of the municipal solid waste material tofermentable sugars and to convert the fermentable sugars into a finalproduct (Lynd et al., 2002, Microbiol. Mol. Biol. Reviews 66: 506-577).It is understood herein that any method known in the art comprisingpretreatment, enzymatic hydrolysis, fermentation, or a combinationthereof, can be used in the practicing the processes of the presentinvention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65).Additional reactor types include fluidized bed, upflow blanket,immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment. In practicing the processes of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of the municipal solid waste material (Chandra et al.,2007, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi,2007, Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman,2009, Bioresource Technology 100: 10-18; Mosier et al., 2005,Bioresource Technology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J.Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

In a preferred embodiment of the invention MSW is subject to a mild tosevere temperature pretreatment in the range 10-300° C. prior tohydrolysis. Heating will normally occur together with a mixing. Heatingwill normally be carried out by addition of water or steam. Pretreatmentmight also consist of a separation (manual or automatic) of MSW indifferent fractions. The municipal solid waste material can also besubjected to particle size reduction, sieving, pre-soaking, wetting,washing, and/or conditioning prior to pretreatment using methods knownin the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The municipal solid waste material can be pretreated before hydrolysisand/or fermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The municipal solid waste material can be pretreated both physically(mechanically) and chemically. Mechanical or physical pretreatment canbe coupled with steaming/steam explosion, hydrothermolysis, dilute ormild acid treatment, high temperature, high pressure treatment,irradiation (e.g., microwave irradiation), or combinations thereof. Inone aspect, high pressure means pressure in the range of preferablyabout 100 to about 400 psi, e.g., about 150 to about 250 psi. In anotheraspect, high temperature means temperature in the range of about 100 toabout 300° C., e.g., about 140 to about 200° C. In a preferred aspect,mechanical or physical pretreatment is performed in a batch-processusing a steam gun hydrolyzer system that uses high pressure and hightemperature as defined above, e.g., a Sunds Hydrolyzer available fromSunds Defibrator AB, Sweden. The physical and chemical pretreatments canbe carried out sequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the municipal solid waste materialis subjected to physical (mechanical) or chemical pretreatment, or anycombination thereof, to promote the separation and/or release ofcellulose, hemicellulose, and/or lignin.

Biological Pretreatment. The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the municipal solidwaste material. Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol.39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass:a review, in Enzymatic Conversion of Biomass for Fuels Production,Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15;Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanolproduction from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Enz.Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

In other embodiments MSW can be pretreated both physically(mechanically) and chemically. Mechanical or physical pretreatment canbe coupled with steaming/steam explosion, hydrothermolysis, dilute ormild acid treatment, high temperature, high pressure treatment,irradiation (e.g., microwave irradiation), or combinations thereof. Inone aspect, high pressure means pressure in the range of preferablyabout 100 to about 400 psi, e.g., about 150 to about 250 psi. In anotheraspect, high temperature means temperature in the range of about 100 toabout 300° C., e.g., about 140 to about 200° C. In a preferred aspect,mechanical or physical pretreatment is performed in a batch-processusing a steam gun hydrolyzer system that uses high pressure and hightemperature as defined above, e.g., a Sunds Hydrolyzer available fromunds Defibrator AB, Sweden. The physical and chemical pretreatments canbe carried out sequentially or simultaneously, as desired.

Hydrolysis. In the hydrolysis step, the municipal solid waste material,e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose and other substrates to fermentable sugars, such asglucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose,and/or soluble oligosaccharides (also known as saccharification). Thehydrolysis is performed enzymatically by one or more enzyme compositionsin one or more stages. In the hydrolysis step, the municipal solid wastematerial, e.g., pretreated, is hydrolyzed to break down proteins andlipids (e.g. triglycerides) found in the waste.

The hydrolysis can be carried out as a batch process or series of batchprocesses. The hydrolysis can be carried out as a fed batch orcontinuous process, or series of fed batch or continuous processes,where the municipal solid waste material is fed gradually to, forexample, a hydrolysis solution containing an enzyme composition. In anembodiment the hydrolysis a continuous hydrolysis in which a MSWmaterial and a enzymes composition are added at different intervalsthroughout the hydrolysis and the hydrolysate is removed at differentintervals throughout the hydrolysis. The removal of the hydrolysate mayoccur prior to, simultaneously with, or after the addition of thecellulosic material and the cellulolytic enzymes composition.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzymes(s), i.e., optimalfor the enzyme(s).

In one aspect, the saccharification is performed in the presence ofdissolved oxygen at a concentration of at least 0.5% of the saturationlevel.

In an embodiment of the invention the dissolved oxygen concentrationduring saccharification is in the range of at least 0.5% up to 30% ofthe saturation level, such as at least 1% up to 25%, at least 1% up to20%, at least 1% up to 15%, at least 1% up to 10%, at least 1% up to 5%,and at least 1% up to 3%. In a preferred embodiment, the dissolvedoxygen concentration is maintained at a concentration of at least 0.5%up to 30% of the saturation level, such as at least 1% up to 25%, atleast 1% up to 20%, at least 1% up to 15%, at least 1% up to 10%, atleast 1% up to 5%, and at least 1% up to 3% during at least 25%, such asat least 50% or at least 75% of the saccharification period. When theenzyme composition comprises an oxidoreductase the dissolved oxygenconcentration may be higher up to 70% of the saturation level.

Oxygen is added to the vessel in order to achieve the desiredconcentration of dissolved oxygen during saccharification. Maintainingthe dissolved oxygen level within a desired range can be accomplished byaeration of the vessel, tank or the like by adding compressed airthrough a diffuser or sparger, or by other known methods of aeration.The aeration rate can be controlled on the basis of feedback from adissolved oxygen sensor placed in the vessel/tank, or the system can runat a constant rate without feedback control. In the case of a hydrolysistrain consisting of a plurality of vessels/tanks connected in series,aeration can be implemented in one or more or all of the vessels/tanks.Oxygen aeration systems are well known in the art. According to theinvention any suitable aeration system may be used. Commercial aerationsystems are designed by, e.g., Chemineer, Derby, England, and build by,e.g., Paul Mueller Company, MO, USA.

The enzyme compositions can comprise any protein useful in degrading themunicipal solid waste material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, an AA9 polypeptide, a hemicellulase, an esterase, anexpansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, aprotease, and a swollenin. In another aspect, the cellulase ispreferably one or more (e.g., several) enzymes selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase. In another aspect, the hemicellulase is preferably oneor more (e.g., several) enzymes selected from the group consisting of anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. In another aspect, theoxidoreductase is preferably one or more (e.g., several) enzymesselected from the group consisting of a catalase, a laccase, and aperoxidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises anAA9 polypeptide. In another aspect, the enzyme composition comprises anendoglucanase and an AA9 polypeptide. In another aspect, the enzymecomposition comprises a cellobiohydrolase and an AA9 polypeptide. Inanother aspect, the enzyme composition comprises a beta-glucosidase andan AA9 polypeptide. In another aspect, the enzyme composition comprisesan endoglucanase and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase I, acellobiohydrolase II, or a combination of a cellobiohydrolase I and acellobiohydrolase II. In another aspect, the enzyme compositioncomprises an endoglucanase and a beta-glucosidase. In another aspect,the enzyme composition comprises a beta-glucosidase and acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, anAA9 polypeptide, and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase, an AA9 polypeptide, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a beta-glucosidase, andan AA9 polypeptide. In another aspect, the enzyme composition comprisesa beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises a beta-glucosidase, anAA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, abeta-glucosidase, and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase, a beta-glucosidase, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a cellobiohydrolase, abeta-glucosidase, and an AA9 polypeptide. In another aspect, the enzymecomposition comprises an endoglucanase, a beta-glucosidase, an AA9polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In anembodiment, the xylanase is a Family 10 xylanase. In another embodiment,the xylanase is a Family 11 xylanase. In another aspect, the enzymecomposition comprises a xylosidase (e.g., beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a ligninolytic enzyme. In anembodiment, the ligninolytic enzyme is a manganese peroxidase. Inanother embodiment, the ligninolytic enzyme is a lignin peroxidase. Inanother embodiment, the ligninolytic enzyme is a H₂O₂-producing enzyme.In another aspect, the enzyme composition comprises a pectinase. Inanother aspect, the enzyme composition comprises an oxidoreductase. Inan embodiment, the oxidoreductase is a catalase. In another embodiment,the oxidoreductase is a laccase. In another embodiment, theoxidoreductase is a peroxidase. In another aspect, the enzymecomposition comprises a protease. In another aspect, the enzymecomposition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may benative proteins, recombinant proteins, or a combination of nativeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. It is understood herein that therecombinant proteins may be heterologous (e.g., foreign) and/or nativeto the host cell. One or more (e.g., several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and polypeptides having enzymaticactivity depend on several factors including, but not limited to, themixture of cellulolytic enzymes and/or hemicellulolytic enzymes, themunicipal solid waste material, the concentration of municipal solidwaste material, the pretreatment(s) of the municipal solid wastematerial, temperature, time, pH, and inclusion of a fermenting organism(e.g., for Simultaneous Saccharification and Fermentation).

In one aspect, an effective amount of the enzyme composition to themunicipal solid waste material is about 0.5 to about 50 mg, e.g., about0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about 20 mg,about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5 toabout 10 mg per g of the municipal solid waste material. In a relatedaspect, the protease is present at a ratio between 0-20% w/w, such ase.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20% w/w of the total enzyme protein.

In one aspect, the beta-glucanase is present at a ratio between 0-30%w/w, such as e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 32, 24, 25, 26, 27, 28, 29 or 30% w/w of thetotal enzyme protein. In a related aspect, the pectate-lyase is presentat a ratio between 0-10% w/w, such as e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10% w/w of the total enzyme protein. In a related aspect, the mannanaseor amylase is present at a ratio between 0-10% w/w, such as e.g. 1, 2,3, 4, 5, 6, 7, 8, 9, 10% w/w of the total enzyme protein. In yet anotherrelated aspect, the cellulolytic enzyme blend is present at a ratiobetween 40%-99% w/w, such as e.g. between 50%-90% w/w, such as e.g.60%-80% w/w, such as e.g. 65-75% of the total enzyme protein.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the municipal solid waste material, e.g., AA9polypeptides can be derived or obtained from any suitable origin,including, archaeal, bacterial, fungal, yeast, plant, or animal origin.The term “obtained” also means herein that the enzyme may have beenproduced recombinantly in a host organism employing methods describedherein, wherein the recombinantly produced enzyme is either native orforeign to the host organism or has a modified amino acid sequence,e.g., having one or more (e.g., several) amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtained by,e.g., site-directed mutagenesis or shuffling.

Each polypeptide may be a bacterial polypeptide. For example, eachpolypeptide may be a Gram-positive bacterial polypeptide having enzymeactivity, or a Gram-negative bacterial polypeptide having enzymeactivity.

Each polypeptide may also be a fungal polypeptide, e.g., a yeastpolypeptide or a filamentous fungal polypeptide.

Chemically modified or protein engineered mutants of polypeptides mayalso be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host can be a heterologous host (enzyme is foreign tohost), but the host may under certain conditions also be a homologoushost (enzyme is native to host). Monocomponent cellulolytic proteins mayalso be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST® (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP(Genencor Int.), ACCELLERASE™ TRIO (DuPont), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), or ALTERNAFUEL®CMAX3™ (Dyadic International, Inc.). The cellulolytic enzyme preparationis added in an amount effective from about 0.001 to about 5.0 wt. % ofsolids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 toabout 2.0 wt. % of solids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655; WO 00/70031; WO05/093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990,Gene 90: 9-14), Thermobifida fusca endoglucanase III (WO 05/093050), andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reeseiendoglucanase II (Saloheimo et al., 1988, Gene 63:11-22), Trichodermareesei Cel5A endoglucanase II (GenBank:M19373), Trichoderma reeseiendoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563, GenBank:AB003694), Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228,GenBank:Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990,Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase(Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusariumoxysporum endoglucanase (GenBank:L29381), Humicola grisea var.thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomycesendoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase(GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthorathermophila CBS 117.65 endoglucanase, Thermoascus aurantiacusendoglucanase I (GenBank:AF487830), Trichoderma reesei strain No.VTT-D-80133 endoglucanase (GenBank:M15665), and Penicillium pinophilumendoglucanase (WO 2012/062220).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Aspergillus fumigatus cellobiohydrolase I (WO2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO2013/028928), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Penicillium occitanis cellobiohydrolase I (GenBank:AY690482),Talaromyces emersonii cellobiohydrolase I (GenBank:AF439936), Thielaviahyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestriscellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

In the processes of the present invention, any AA9 polypeptide can beused as a component of the enzyme composition.

Examples of AA9 polypeptides useful in the processes of the presentinvention include, but are not limited to, AA9 polypeptides fromThielavia terrestris (WO 2005/074647, WO 2008/148131, and WO2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868, and WO 2009/033071), Aspergillus fumigatus(WO 2010/138754), Penicillium pinophilum (WO 2011/005867), Thermoascussp. (WO 2011/039319), Penicillium sp. (emersoni0 (WO 2011/041397 and WO2012/000892), Thermoascus crustaceous (WO 2011/041504), Aspergillusaculeatus (WO 2012/125925), Thermomyces lanuginosus (WO 2012/113340, WO2012/129699, WO 2012/130964, and WO 2012/129699), Aurantiporusalborubescens (WO 2012/122477), Trichophaea saccata (WO 2012/122477),Penicillium thomii (WO 2012/122477), Talaromyces stipitatus (WO2012/135659), Humicola insolens (WO 2012/146171), Malbranchea cinnamomea(WO 2012/101206), Talaromyces leycettanus (WO 2012/101206), andChaetomium thermophilum (WO 2012/101206), and Talaromyces thermophilus(WO 2012/129697 and WO 2012/130950).

In one aspect, the AA9 polypeptide is used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043, e.g.,manganese or copper.

In another aspect, the AA9 polypeptide is used in the presence of adioxy compound, a bicyclic compound, a heterocyclic compound, anitrogen-containing compound, a quinone compound, a sulfur-containingcompound, or a liquor obtained from a pretreated municipal solid wastematerial such as pretreated corn stover (WO 2012/021394, WO 2012/021395,WO 2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401, WO2012/021408, and WO 2012/021410).

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described in WO2012/021401, and the soluble contents thereof. A liquor for cellulolyticenhancement of an AA9 polypeptide can be produced by treating alignocellulose or hemicellulose material (or feedstock) by applying heatand/or pressure, optionally in the presence of a catalyst, e.g., acid,optionally in the presence of an organic solvent, and optionally incombination with physical disruption of the material, and thenseparating the solution from the residual solids. Such conditionsdetermine the degree of cellulolytic enhancement obtainable through thecombination of liquor and an AA9 polypeptide during hydrolysis of acellulosic substrate by a cellulolytic enzyme preparation. The liquorcan be separated from the treated material using a method standard inthe art, such as filtration, sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), andALTERNA FUEL 200P (Dyadic).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thermomyces lanuginosus (GeneSeqP:BAA22485),Talaromyces thermophilus (GeneSeqP:BAA22834), Thielavia terrestris NRRL8126 (WO 2009/079210), and Trichophaea saccata (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt:Q7SOW4), Trichoderma reesei(UniProtKB/TrEMBL:Q92458), Talaromyces emersonii (SwissProt:Q8X212), andTalaromyces thermophilus (GeneSeqP:BAA22816).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum(UniProt:Q2GWX4), Chaetomium gracile (GeneSeqP:AAB82124), Humicolainsolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036),Myceliophtera thermophila (WO 2010/014880), Neurospora crassa(UniProt:q7s259), Phaeosphaeria nodorum (UniProt:QOUHJ1), and Thielaviaterrestris NRRL 8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt:A1 D9T4), Neurospora crassa(UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), andThielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt:alcc12), Aspergillus fumigatus(SwissProt:Q4WW45), Aspergillus niger (UniProt:Q96WX9), Aspergillusterreus (SwissProt:Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt:Q8X211), and Trichoderma reesei (UniProt:Q99024).

Examples of oxidoreductases useful in the processes of the presentinvention include, but are not limited to, Aspergillus lentiluscatalase, Aspergillus fumigatus catalase, Aspergillus niger catalase,Aspergillus oryzae catalase, Humicola insolens catalase, Neurosporacrassa catalase, Penicillium emersonii catalase, Scytalidiumthermophilum catalase, Talaromyces stipitatus catalase, Thermoascusaurantiacus catalase, Coprinus cinereus laccase, Myceliophthorathermophila laccase, Polyporus pinsitus laccase, Pycnoporus cinnabarinuslaccase, Rhizoctonia solani laccase, Streptomyces coelicolor laccase,Coprinus cinereus peroxidase, Soy peroxidase, Royal palm peroxidase.

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

In preferred embodiments, some fermentation will occur concurrent withthe hydrolysis of the MSW. Fermentable sugars obtained from thehydrolyzed municipal solid waste material can be fermented by one ormore (e.g., several) fermenting microorganisms capable of fermenting thesugars directly or indirectly into a fermentation products such asvolatile fatty acids (e.g. acetate, propionate, butyrate), lactate andalcohols.

“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. Fermentationprocesses also include fermentation processes used in the consumablealcohol industry (e.g., beer and wine), dairy industry (e.g., fermenteddairy products), leather industry, and tobacco industry. Thefermentation conditions depend on the desired fermentation product andfermenting organism and can easily be determined by one skilled in theart.

In the fermentation step, sugars, released from the municipal solidwaste material as a result of the pretreatment and enzymatic hydrolysissteps, are fermented to a product, e.g., ethanol, by a fermentingorganism, such as yeast. Hydrolysis and fermentation can be separate orsimultaneous.

Any suitable hydrolyzed municipal solid waste material can be used inthe fermentation step in practicing the present invention. The materialis generally selected based on economics, i.e., costs per equivalentsugar potential, and recalcitrance to enzymatic conversion.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Yeast includestrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Xylose fermenting yeast include strains of Candida, preferably C.sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, suchas P. stipitis CBS 5773. Pentose fermenting yeast include strains ofPachysolen, preferably P. tannophilus. Organisms not capable offermenting pentose sugars, such as xylose and arabinose, may begenetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In an aspect, the fermenting microorganism has been genetically modifiedto provide the ability to ferment pentose sugars, such as xyloseutilizing, arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

In another aspect, the fermenting organism comprises one or morepolynucleotides encoding one or more cellulolytic enzymes,hemicellulolytic enzymes, and accessory enzymes described herein.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedmunicipal solid waste material or hydrolysate and the fermentation isperformed for about 8 to about 96 hours, e.g., about 24 to about 60hours. The temperature is typically between about 26° C. to about 60°C., e.g., about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH4-5, 6, or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded municipal solid waste material and the fermentation isperformed for about 12 to about 96 hours, such as typically 24-60 hours.In another aspect, the temperature is preferably between about 20° C. toabout 60° C., e.g., about 25° C. to about 50° C., about 32° C. to about50° C., or about 32° C. to about 50° C., and the pH is generally fromabout pH 3 to about pH 7, e.g., about pH 4 to about pH 7. However, somefermenting organisms, e.g., bacteria, have higher fermentationtemperature optima. Yeast or another microorganism is preferably appliedin amounts of approximately 10⁵ to 10¹², preferably from approximately10⁷ to 10¹⁰, especially approximately 2×10⁸ viable cell count per ml offermentation broth. Further guidance in respect of using yeast forfermentation can be found in, e.g., “The Alcohol Textbook” (Editors K.Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University Press,United Kingdom 1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g., pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide.

In one aspect, the fermentation product is an alcohol. The term“alcohol” encompasses a substance that contains one or more hydroxylmoieties. The alcohol can be, but is not limited to, n-butanol,isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol,glycerin, glycerol, 1,3-propanediol, sorbitol, xylitol. See, forexample, Gong et al., 1999, Ethanol production from renewable resources,in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira andJonas, 2002, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh,1995, Process Biochemistry 30(2): 117-124; Ezeji et al., 2003, WorldJournal of Microbiology and Biotechnology 19(6): 595-603.

In another aspect, the fermentation product is an alkane. The alkane maybe an unbranched or a branched alkane. The alkane can be, but is notlimited to, pentane, hexane, heptane, octane, nonane, decane, undecane,or dodecane.

In another aspect, the fermentation product is a cycloalkane. Thecycloalkane can be, but is not limited to, cyclopentane, cyclohexane,cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene maybe an unbranched or a branched alkene. The alkene can be, but is notlimited to, pentene, hexene, heptene, or octene.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide),cell debris, biomass, fermentation media and/or fermentation products.In some embodiments, the composition is a cell-killed whole brothcontaining organic acid(s), killed cells and/or cell debris, and culturemedium.

The term “fermentation broth” refers to a preparation produced bycellular fermentation that undergoes no or minimal recovery and/orpurification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, ahemicellulase, a cellulose inducible protein (CIP), an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin. The fermentation broth formulations or cellcompositions may also comprise one or more (e.g., several) enzymesselected from the group consisting of a hydrolase, an isomerase, aligase, a lyase, an oxidoreductase, or a transferase, e.g., analpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, glucoamylase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of uses of the compositions of the presentinvention. The dosage of the composition and other conditions underwhich the composition is used may be determined on the basis of methodsknown in the art.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellobiohydrolase activity of the composition has been increased, e.g.,with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, an AA9polypeptide, a CIP, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Thecompositions may also comprise one or more (e.g., several) enzymesselected from the group consisting of a hydrolase, an isomerase, aligase, a lyase, an oxidoreductase, or a transferase, e.g., analpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, glucoamylase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, or xylanase.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

In yet further embodiments, the enzymes of the composition of presentinvention may be a protease derived from Bacillus amyloliquefaciens, atriacylglycerol lipase with a phospho-lipase activity derived fromThermomyces lanuginosus, a triacylglycerol lipase derived from Humicolainsolens, a triacylglycerol lipase derived from Thermomyces lanuginosus,NZ81243—a multicomponent enzyme commercially available as Novozym 81243with pectate lyase, xylanase and cellulase activities, a beta-glucanasederived from Aspergillus aculeatus. an alpha-amylase derived fromRhizomucor pusillus and/or an endo-mannosidase derived from Bacillusbogoriensis.

Examples are given below of uses of the compositions of the presentinvention. The dosage of the composition and other conditions underwhich the composition is used may be determined on the basis of methodsknown in the art.

Materials and Methods

1. Substrate Preparation

Unsorted MSW is basically made up of two solid fractions: 1) Inorganicnon-degradables consisting of plastic, glass, metal etc. and 2)Degradable organics consisting of vegetable, animal waste, food waste,paper, cardboard etc. As an example the latter part typically makes upapproximately 65-70% of the incoming waste in Denmark (for a detailedcomposition analysis see Riber et al. 2009, Waste Management 29, p.1251-1257).

The scope of the experiments described below was to optimize thesolubilization of the degradable organic parts of MSW. Therefore, wechoose to use a model waste that reflected the composition of degradableorganics in MSW as described by Riber et al. 2009. The model wasteconsisted of 3 fractions based on fresh products supplemented withwater:

-   -   40.1% Vegetable origin. Mix of fresh vegetables (onions,        cabbage, carrots, cucumber etc.), cereals (oatmeals, corn flakes        etc.), bread, cake, flowers, boiled rice, boiled pasta fruit,        ketchup etc.    -   11.3% Animal origin. Mix of pâté, sausage, hotwings, spareribs,        crude meats from chicken, pork and beef etc.    -   36.2% Cellulose origin. Mix of newsprints, office paper,        magazines, cardboard, juice cartons, kitchen tissue, cotton,        wood, textiles etc.    -   Water to obtain a consistency of the model waste suitable in the        assays (described later).

A total of 3 different batches were used prepared and used for theexperiments. Batch 1 and 2 was prepared by REnescience (DONG NS). Batch3 was prepared in our own laboratories. The composition of the modelwaste is given in the table below:

Total solids/dry matter (TS) 25.2-28.3% TS composition: Klason lignin 12% Cellulose 29-32% Xylan  10% Arabinan   1% Galactan 1.7% Mannnan  4% Fats   7% Protein 7-8% Starch  3-10% Ash  15%

2. Assay for Enzymatic Solubilization of Model Waste in Bench Scale.

If not otherwise stated, experiments were carried out on a 20 g scale. Astandard assay for enzymatic solubilization of model waste was used asfollows:

-   1. 50 ml centrifuge tubes+lid were weighed.-   2. Model waste was added to the tubes (total of 1.5 g TS).-   3. 50 mM citric acid buffer (pH 5) (app. 14.7 ml) and enzymes was    added (total weight=20 g and 7.5% TS) and tubes was vigorously    shaken.-   4. Tubes were incubated on a Stuart Rotator SB3 at 12 rpm in ovens    at 50° C. for 24 hours.-   5. After incubation, 0.4 ml 10% proxel was added to kill lactic acid    bacteria and other microorganisms.-   6. The tubes were centrifuged 10 min at 2090×g.-   7. A new set of 50 ml tubes were weighed but without lids, marked as    supernatant.-   8. Supernatant was poured in the tubes and the tubes+sup was    weighed.-   9. Approx. 2×1.5 ml of supernatant was removed and poured in    eppendorf tubes for analysis. 50 ml tubes with supernatant were    weighed again.-   11. The first tube+pellet were weighed again.-   12. Eppendorf tubes with supernatant were stored in freezer.-   13. TS of the supernatant and pellet were determined by drying the    tubes at 50° C. The tubes was weighed and then dried at 105° C. and    weighed again.

Analysis: Mass balances based on weight were made before and afterincubation. If more than 5% of the substrate had been lost samples werediscarded. The ratio of the dry matter content in the supernatant andpellet following centrifugation was calculated for all samples. Ifneeded, samples that had been frozen were analyzed for sugar content,acetic acid and lactate on HPLC.

EXAMPLES Example 1 Screening of Individual Enzymes and Selection ofEnzyme Candidates for Blending.

Initially, a very broad range of enzymes (>50; Novozymes A/S, Bagsværd,Denmark) were screened for investigating the potential of improving thehydrolysis of the model waste. The tested enzymes includedalpha-amylases, gluco-amylase, pullulanase, proteases, lipases,cellulases, xylanases, pectinases and beta-glucanases.

The described standard assay was used. Control vials were added CBC(CELLIC® Ctec3; Novozymes A/S, Bagsværd, Denmark) in an amount of2.4%/TS (product to dry matter). In test vials part of CBC was replacedwith other enzymes on a protein:protein basis in different ratiosranging from 1-50%. Density of CBC and different products was taken intoaccount. Furthermore, blanks with substrate and buffer but no enzymeswere prepared. All tests were carried out in at least duplicates.

The primary success criteria for an improved hydrolysis were defined asan increase in soluble total solids (TS in supernatant). The ratio ofTS-solubilisation during the screening procedure was in general around25-28% for samples with CBC but variations were seen. TheTS-solubilisation of tubes without enzymes was consistent around 10-11%.Candidates were selected for further testing:

-   -   B.a protease (SEQ ID NO: 1): A protease derived from Bacillus        amyloliquefaciens that gave up to 5% increase in        TS-solubilization at 30% replacement of CBC.    -   T.l pholip (SEQ ID NO: 2): A triacylglycerol lipase with        phospho-lipase activity derived from Thermomyces lanuginosus        that gave up to 4.4-8.5% increase in TS-solubilization at 2-20%        replacement of CBC.    -   H.i trilip (H.i trilip): A triacylglycerol lipase derived from        Humicola insolens that gave up to 4-7% increase in        TS-solubilization at 5-20% replacement of CBC.    -   T.l trilip (SEQ ID NO: 3): A triacylglycerol lipase derived from        Thermomyces lanuginosus that gave up to 10-15% increase in        TS-solubilization at 1-10% replacement of CBC.    -   NZ81243 A multicomponent enzyme commercially available as        Novozym 81243 with pectate lyase, xylanase and cellulase        activities. Gave up to 4% increase in TS solubilization at 5-20%        replacement of CBC. 2:1    -   A.a BG: (SEQ ID NO: 4): A beta-glucanase derived from        Aspergillus aculeatus. Contains side activities (cellulase,        xylanases, pectinase). Gave up to 6.2-8.2% increase in TS        solubilization at 20-40% replacement of CBC. Gave up to 16% in        TS solubilization at 30% replacement of CBC when mixed with        NZ81243 in a 2:1 ratio.    -   R.p Alam (SEQ ID NO: 5): A alpha-amylase derived from Rhizomucor        pusillus that improve glucose yields in the supernatant with up        to 20% at 5-20% replacement of CBC.    -   B.b Enma: An endo-mannosidase derived from Bacillus bogoriensis.        Gave up to 6-10% increase in TS solubilization at 2.5-10%        replacement of CBC.

Example 2 Designing of Optimized Enzymes Mix by Blending of SelectedCandidates

Statistical experiments were setup to find the optimal ratio between CBCand the selected enzymes candidates in a multicomponent enzymes blend.Based on the screening experiments we decided to use two differenttemplates that specified the enzymes ratios to be used:

For both templates it was decided to use 0-20% B.a protease (protease)and 0-10% lipase. This was supplemented with either a) 0-30% A.a BG(beta-glucanase)+/−NZ81243 (pectate lyase) or b) 0-20% B.b Enma(endo-mannosidase) or 0-20% R.p Alam (alpha-amylase). A total of 6blending experiments were performed.

Experiment a.1-a.3.

The initial three experiments were carried out to select the mostsuitable lipase when combined with 0-20% B.a protease and 0-30% A.a BG.The templates were as follows:

Blend Template a.1

Enzyme Name Ratio of total enzyme protein: B.a protease 0-20% T.I pholip0-10% A.a BG 0-30% CBC Supplement to 100%

Blend Template a.2

Enzyme Name Ratio of total enzyme protein: B.a protease 0-20% T.I trilip0-10% A.a BG 0-30% CBC Supplement to 100%

Blend Template a.3

Enzyme Name Ratio of total enzyme protein: B.a protease 0-20% H.i trilip0-10% A.a BG 0-30% CBC Supplement to 100%

Statistical discovery software (Design Of Experiments, DOE) by JMP® wasused to design the dosing in the experiment (see table below). As inExample 1, the enzymes concentration in control vials (vial 17 and 39)was 2.4% CBC/TS. In test vials part of CBC was replaced with otherenzymes on a protein:protein basis as stated in the table below. Eachtest blend was carried out in duplicates.

Enzymes Dosing in Tubes. Used for Template a.1, a.2, a.3. a.4. a.5.

T.I pholip or T.I trilip or Tube CBC B.a protease H.i trilip A.a BG #Ratio ( % of enzyme protein) 12 0.4 0.2 0.1 0.3 22 0.4 0.2 0.1 0.3 6 0.50.2 0 0.3 19 0.5 0.1 0.1 0.3 21 0.5 0.1 0.1 0.3 25 0.5 0.2 0 0.3 10 0.550.2 0.1 0.15 26 0.55 0.2 0.1 0.15 9 0.6 0 0.1 0.3 13 0.6 0.1 0 0.3 290.6 0.1 0 0.3 36 0.6 0 0.1 0.3 3 0.65 0 0.05 0.3 4 0.65 0.2 0 0.15 280.65 0 0.05 0.3 33 0.65 0.2 0 0.15 2 0.7 0.2 0.1 0 5 0.7 0.1 0.05 0.1515 0.7 0 0 0.3 23 0.7 0.1 0.05 0.15 24 0.7 0.2 0.1 0 35 0.7 0 0 0.3 70.75 0.2 0.05 0 16 0.75 0 0.1 0.15 30 0.75 0 0.1 0.15 31 0.75 0.2 0.05 08 0.8 0.2 0 0 11 0.8 0.1 0.1 0 37 0.8 0.2 0 0 38 0.8 0.1 0.1 0 14 0.85 00 0.15 34 0.85 0 0 0.15 18 0.9 0 0.1 0 20 0.9 0.1 0 0 27 0.9 0 0.1 0 320.9 0.1 0 0 1 0.95 0 0.05 0 40 0.95 0 0.05 0 17 1 0 0 0 39 1 0 0 0The highlights of test a.1, a.2 and a.3 are given in the tables below:

Results Highlights Blend a.1

B.a protease, T.I pholip, Enzyme components A.a BG, CBC Result control(TS solubilization) 25.8% 1. best blend (TS solubilization) 32.8%Improvement 27.1% Best ratio between enzymes 10:5:15:70 2. best blend(TS solubilization) 30.8% Improvement 19.4% Best ratio between enzymes10:10:0:80 Best ratio according to model 13:7:11:69

Results Highlights Blend a.2

B.a protease, T.I trilip, Enzyme components A.a BG, CBC Result control(TS solubilization) 28.1% 1. best blend (TS solubilization) 34.7%Improvement 23.5% Best ratio between enzymes 20:10:30:40 2. best blend(TS solubilization) 33.4% Improvement 18.9% Best ratio between enzymes10:10:0:80

Results Highlights Blend a.3

B.a protease, H.i trilip, Enzyme components A.a BG, CBC Result control(TS solubilization) 26.1% 1. best blend (TS solubilization) 32.5%Improvement 24.5% Best ratio between enzymes 10:5:15:70 2. best blend(TS solubilization) 31.9% Improvement 22.2% Best ratio between enzymes20:5:0:80

As shown, the best improvement over CBC in TS-solubilization was foundin experiment a.1 with a 27.1% increase with a ratio of 10:5:15:70 forB.a protease:T.l pholip:A.a BG:CBC. Interestingly, the TS-solubilizationin the individual vials showed significant deviations ranging from aslight negative impact by the enzymes mix to a pronounced boost of+30.4% in tube number 23. As written in Example 1, the individualimprovements of B.a protease, T.l pholip and A.a BG was up to 5%, 8.5%and 8.2% respectively. Multiplication of the individual effects onlyadds up to 21.7%. Thus, a synergistic effect was clearly obtained inexperiment a.1 when mixing the enzymes.

Clear improvements in TS solubilization was also seen in experiment a.2(up to 23.5% with a combination of B.a protease:T.l trilip:A.a BG:CBC ina ratio of 20:10:30:40) and a.3 (up to 24.5% with a combination of B.aprotease:H.i trilip:A.a BG:CBC in a ratio of 10:5:15:70). It was howeverdecided to continue with T.l pholip in the following experiments.

Experiment a.4.

This experiment was used to decide whether A.a BG should be supplementedwith NZ81243. The initial screening (Example 1) had shown a boost in TSsolubilization when the two enzymes was combined.

Blend Template a.4

Enzyme Name Ratio of total enzyme protein: B.a protease 0-20% T.I pholip0-10% A.a BG + NZ81243 0-30% (2:1 mix) CBC Supplement to 100%

As shown in the table below, TS-solubilization could be increased to22.1%. However, this was a smaller improvement than in experiment a.1where A.a BG was not supplemented with NZ81243.

Results Highlights Blend a.4

B.a protease, T.I pholip, Enzyme components A.a BG + NZ81243, CBC Resultcontrol (TS solubilization) 26.6% 1. best blend (TS solubilization)32.5% Improvement 22.1% Best ratio between enzymes 0:5:30:65 2. bestblend (TS solubilization) 32.3% Improvement 21.4% Best ratio betweenenzymes 20:5:0:75

Experiment b.1. and b.2.

This experiment was used to decide whether the alpha amylase R.p Alam orendo-mannosidase B.b Enma where a better “match” for the protease B.aprotease and the lipase T.l pholip than A.a BG.

The following blend and dosing templates were used:

Blend Template b.1

Enzyme Name Ratio of total enzyme protein: B.a protease 0-20% T.I pholip0-10% B.b Enma 0-10% CBC Supplement to 100%

Blend Template b.2

Enzyme Name Ratio of total enzyme protein: B.a protease 0-20% T.I pholip0-10% R.p Alam 0-10% CBC Supplement to 100%Enzymes Dosing in Tubes. Used for Template b.1, b.2.

B.a T.I B.b Enma or Tube CBC protease pholip R.p Alam # Ratio ( % ofenzyme protein) 15 0.6 0.2 0.1 0.1 37 0.6 0.2 0.1 0.1 10 0.65 0.2 0.050.1 17 0.65 0.2 0.1 0.05 27 0.65 0.2 0.1 0.05 40 0.65 0.2 0.05 0.1 3 0.70.1 0.1 0.1 5 0.7 0.2 0 0.1 8 0.7 0.2 0.1 0 29 0.7 0.2 0 0.1 30 0.7 0.10.1 0.1 35 0.7 0.2 0.1 0 16 0.75 0.2 0.05 0 20 0.75 0.2 0 0.05 26 0.750.2 0.05 0 28 0.75 0.2 0 0.05 13 0.8 0.1 0.1 0 14 0.8 0 0.1 0.1 18 0.80.1 0.05 0.05 19 0.8 0.2 0 0 31 0.8 0.2 0 0 33 0.8 0.1 0.1 0 34 0.8 00.1 0.1 38 0.8 0.1 0.05 0.05 4 0.85 0 0.05 0.1 12 0.85 0 0.1 0.05 320.85 0 0.05 0.1 39 0.85 0 0.1 0.05 2 0.9 0 0 0.1 6 0.9 0 0.1 0 9 0.9 0.10 0 21 0.9 0 0.1 0 24 0.9 0 0 0.1 25 0.9 0.1 0 0 1 0.95 0 0 0.05 7 0.950 0.05 0 22 0.95 0 0.05 0 23 0.95 0 0 0.05 11 1 0 0 0 36 1 0 0 0

Results Highlights Blend b.1

B.a protease, T.I pholip, Enzyme components B.b Enma, CBC Result control(TS solubilization) 27.4% 1. best blend (TS solubilization) 34.0%Improvement 24.1% Best ratio between enzymes 20:0:30:50 2. best blend(TS solubilization) 33.1% Improvement 20.8% Best ratio between enzymes20:5:10:65

Results Highlights Blend b.2

B.a protease, T.I pholip, Enzyme components R.p Alam, CBC Result control(TS solubilization) 25.7% 1. best blend (TS solubilization) 30.7%Improvement 19.4% Best ratio between enzymes  0:5:30:60 2. best blend(TS solubilization) 30.3% Improvement 17.9% Best ratio between enzymes20:5:0:75

In both blending experiments an improved performance inTS-solubilization was found when compared to CBC, but all test wereinferior to the blend ratio found in experiment a.1 where B.a protease,T.l pholip, A.a BG, where mixed with CBC in a ratio of 10:5:15:70.

Based on these observations it was decided to use this enzymescombination in the further development.

Example 3 Testing the Selected Enzymes Blend and CELLIC® Ctec3 in FreeFall Experiments at Elevated Dry Matter Concentration.

An experiment was carried out to test the efficiency of the selectedenzymes blend at a dry matter concentration that was higher than duringthe screening and blending experiments. However, due to a high viscosityof model waste, mixing is not optimal in 50 ml tubes when experimentsare carried at dry matter concentrations above 7.5%. Instead,experiments were performed in 100 ml Kautex bottles. Model waste wasmixed with water to a volume of 50 ml and at TS concentration of 7.5%and 15%. CBC and the selected blend (B.a protease:T.l pholip:A.a BG:CBCin ratio of 10:5:15:70) were added in an amount of 2.4%/TS (product todry matter CBC). Tubes were incubated for 24 hours at 50° C. in thetumbler-reactor. Results are shown in FIG. 1.

TS solubilization of original biomass in control tubes (no enzymes) were10.9% and 3.2% at 7.5% and 15%, respectively. Addition of CBC improvedTS-solubilization to 29.6% and 25.8. Supplementing CBC with the otherenzymes improved solubilization further to 37.1 and 30.7%. Thiscorresponds to a relative improvement of 25.7% (7.5% dry matter) and18.9% (15% dry matter), when comparing the blend with CBC. The numbersobtained at 7.5% dry matter confirms the improvement seen in 20 g scale(50 ml tubes), which was up to 27.3%.

Example 4. Comparable Dose-Response Experiments with CBC and the EnzymesBlend

Experiments were performed in 100 ml Kautex bottles. Model waste wasmixed with water to a volume at 50 ml and at TS concentration of 7.5%.CBC and the selected blend (B.a protease:T.l pholip:A.a BG:CBC in ratioof 10:5:15:70) were added in amounts corresponding to 0%, 25%, 50%, 75%,100% and 200% of the concentration that has been used as default duringthe previous experiments (2.4% enzymes protein/TS). Bottles wereincubated on a Stuart Rotator SB3 and placed in a 50° C. oven for 24hours.

A significant improvement in TS-solubilization was seen at all appliedenzyme concentrations, when comparing the blend with CBC. TheTS-solubilization at default settings (2.4% CBC/TS) was around 25%. Thiswas obtained with only approximately 0.9% of the blend, whichcorresponds to a lowering in enzyme dosage of approximately 2.5 to 2.7times (See FIG. 2). At the same time we found a clear increase inhydrolysis and fermentation products such as glucose, xylose, lacticacid (FIG. 3, and FIG. 5). This is a surprise since 15% of CBC(cellulase and xylanase activities) was replaced with the lipase andprotease.

Example 5

Omitting Individual Components from Blend

An experiment was carried out to test the relevance of the individualenzymes in the optimized blend (B.a protease:T.l pholip:A.a BG:CBC inratio of 10:5:15:70). The setup of control vials was as described inExample 1 (2.4% CELLIC® Ctec, 7.5% TS, 20 gram scale). In test vials theoptimized blend was applied, including all enzymes. At the same timetest vials were made were either B.a protease, T.l pholip or A.a BG hadbeen excluded from the blend.

The effect on TS-solubilization is illustrated in FIG. 4 and clearlyshows that removing any of the individual enzymes resulted in a lowerTS-solubilization, when compared to vials with all enzymes. However,when only using two (2) of the selected enzymes to supplement CBC (T.lpholip+A.a BG, B.a protease+A.a BG, B.a protease+T.l pholip) we stillobserved an improved TS-solubilization, when compared to vials with onlyCBC, even though total enzymes protein concentration had been lowered byremoving individual enzymes.

Although the foregoing has been described in some detail by way ofillustration and example for the purposes of clarity of understanding,it is apparent to those skilled in the art that any equivalent aspect ormodification, may be practiced. Therefore, the description and examplesshould not be construed as limiting the scope of the invention.

The present invention may be further described by the following numberedparagraphs:[1] An enzyme composition for solubilization of Municipal Solid Waste(MSW), the enzyme composition comprising: (i) a cellulolytic backgroundcomposition and (ii) a protease; and/or (iii) a lipase.[2] The composition of paragraph [1], further comprising (iv) abeta-glucanase; (v) a pectate lyase; (vi) a mannanase and/or (vii) anamylase.[3] The composition of paragraph [1] or [2], wherein the cellulolyticbackground composition comprises a) a cellobiohydrolase I or variantthereof; (b) cellobiohydrolase II or variant thereof; (c)beta-glucosidase or variant thereof; and (d) a polypeptide havingcellulolytic enhancing activity; or homologs thereof.[4] The composition of any of paragraphs [1] to [3], wherein thecellulolytic background composition comprise (a) an Aspergillusfumigatus cellobiohydrolase I or variant thereof; (b) an Aspergillusfumigatus cellobiohydrolase II or variant thereof; (c) an Aspergillusfumigatus beta-glucosidase or variant thereof; and (d) a Penicillium sp.GH61 polypeptide having cellulolytic enhancing activity; or homologsthereof.[5] The composition of any of paragraphs [1] to [4], wherein the (ii) aprotease is derived from the genus Bacillus, such as e.g. Bacillusamyloliquefaciens such as e.g. the protease encoded by SEQ ID NO: 1.[6] The composition of any of paragraphs [1] to [5], wherein the (iii) alipase is derived from the genus Thermomyces sp. such as e.g.Thermomyces lanuginosus such as e.g. the lipase encoded by SEQ ID NO: 2or wherein the (ii) a lipase is derived from the genus Humicola sp. suchas e.g. Humicola insolens.[7] The composition of any of paragraphs [1] to [6], wherein the (iv) abeta-glucanase is derived from a member of the genus Aspergillus such ase.g. Aspergillus aculeatus such as e.g. the beta-glucanase encoded bythe sequence encoded by SEQ ID NO: 4 or homologs thereof.[8] The composition of any of paragraphs [1] to [7], wherein the (v) apectate lyase forms part of a multicomponent enzyme compositioncomprising pectate lyase, xylanase and cellulase activities such as e.g.Novozym 81243™.[9] The composition of any of paragraphs [1] to [8], wherein the (vi) amannanase is an endo-mannosidase derived from the genus Bacillus such ase.g. Bacillus bogoriensis such as e.g. the endo-mannosidase encoded bySEQ ID NO: 6 or homologs thereof.[10] The composition of any of paragraphs [1] to [9], wherein the (vii)an amylase is an alpha-amylase derived from the genus Rhozimucor such ase.g. Rhizomucor pusillus such as e.g. the alpha-amylase encoded by SEQID NO: 5 or homologs thereof.[11] The composition of any of paragraphs [1] to [10], wherein theprotease is present at a ratio between 0-20% w/w, such as e.g. 10% w/wof the total enzyme protein.[12] The composition of any of paragraphs [1] to [11], wherein thebeta-glucanase is present at a ratio between 0-30% w/w, such as e.g. 15%w/w of the total enzyme protein.[13] The composition of any of paragraphs [1] to [12], wherein thepectate-lyase is present at a ratio between 0-10% w/w, such as e.g. 5%w/w of the total enzyme protein.[14] The composition of any of paragraphs [1] to [13], wherein themannanase or amylase is present at a ratio between 0-10% w/w, such ase.g. 5% w/w of the total enzyme protein.[15] The composition of any of paragraphs [1] to [14], wherein thecellulolytic enzyme blend is present at a ratio between 40%-99% w/w,such as e.g. between 50%-90% w/w, such as e.g. 60%-80% w/w, such as e.g.65-75% of the total enzyme protein.[16] The composition of any of paragraphs [1] to [15], wherein theenzyme composition further comprises one or more enzymes selected fromthe group consisting of a cellulase, an AA9 polypeptide, ahemicellulase, a cellulose inducible protein (CIP) an esterase, anexpansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, aprotease, and a swollenin.[17] The composition of any of paragraphs [1] to [16], wherein thehemicellulase is one or more enzymes selected from the group consistingof a xylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.[18] A process for solubilizing waste comprising:

(a) contacting waste with the enzyme composition of any of paragraphs[1]-[17].

[19] The process of paragraph [18], wherein the waste is Municipal SolidWaste (MSW).[20] A process for producing a fermentation product, comprising:

(a) treating MSW with the enzyme composition of any of paragraphs[1]-[17]

(b) fermenting the solubilized and/or hydrolysed MSW with one or morefermenting microorganisms to produce a fermentation product; and

(c) recovering the fermentation product from the fermentation.

[21] The process of any of paragraphs [18]-[20], wherein the waste ispretreated.

What is claimed is:
 1. A process for solubilizing waste comprising: (a)contacting waste with an enzyme composition comprising a cellulolyticbackground composition and one or more enzymes selected from (i) aprotease; (ii) a lipase and (iii) a beta-glucanase.
 2. The process ofclaim 1, wherein the waste is Municipal Solid Waste (MSW).
 3. A processfor producing a fermentation product, comprising: (a) treating MunicipalSolid Waste (MSW) with an enzyme composition comprising a cellulolyticbackground composition and one or more enzymes selected from (i) aprotease; (ii) a lipase and (iii) a beta-glucanase; (b) fermenting thesolubilized and/or hydrolysed MSW with one or more fermentingmicroorganisms to produce a fermentation product; and (c) recovering thefermentation product from the fermentation.
 4. The process of any ofclaims 1-3, wherein the waste is pretreated.
 5. The process of any ofclaims 1-4, wherein the composition comprises two or more enzymesselected from (i) a protease; (ii) a lipase and (iii) a beta-glucanase(e.g., a protease and a lipase; a protease and a beta-glucanase; or alipase and a beta-glucanase).
 6. The process of any of claims 1-4,wherein the enzyme composition comprises (i) a protease; (ii) a lipaseand (iii) a beta-glucanase.
 7. The process of any of claims 1-6, whereinthe enzyme composition further comprises one or more enzymes selectedfrom (iv) a pectate lyase; (v) a mannanase; and (vi) an amylase.
 8. Theprocess of any of claims 1-7, wherein the cellulolytic backgroundcomposition comprises one or more enzymes selected from (a) acellobiohydrolase I or variant thereof; (b) cellobiohydrolase II orvariant thereof; (c) beta-glucosidase or variant thereof; and (d) apolypeptide having cellulolytic enhancing activity; or homologs thereof.9. The process of any of claims 1-7, wherein the cellulolytic backgroundcomposition comprises (a) a cellobiohydrolase I or variant thereof; (b)cellobiohydrolase II or variant thereof; (c) beta-glucosidase or variantthereof; and (d) a polypeptide having cellulolytic enhancing activity;or homologs thereof.
 10. The process of claim 8 or 9, wherein thecellobiohydrolase I of (a) is an Aspergillus fumigatus cellobiohydrolaseI or variant thereof; the cellobiohydrolase II of (b) is an Aspergillusfumigatus cellobiohydrolase II or variant thereof; the beta-glucosidaseof (c) is an Aspergillus fumigatus beta-glucosidase or variant thereof;and the polypeptide having cellulolytic enhancing activity of (d) is aPenicillium sp. GH61 polypeptide having cellulolytic enhancing activity;or homologs thereof.
 11. The process of any of claims 1 to 10, whereinthe protease (i) of the enzyme composition is derived from the genusBacillus, such as e.g. Bacillus amyloliquefaciens such as e.g. theprotease encoded by SEQ ID NO: 1 (e.g., a protease having at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 1).
 12. The process of any ofclaims 1 to 11, wherein the lipase (ii) of the enzyme composition isderived from the genus Thermomyces sp. such as e.g. Thermomyceslanuginosus such as e.g. the lipase encoded by SEQ ID NO: 2 (e.g., alipase having at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2) orwherein the lipase (ii) is derived from the genus Humicola sp. such ase.g. Humicola insolens (e.g., a lipase having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the Humicola insolens lipase).
 13. The process ofany of claims 1 to 12, wherein the beta-glucanase (iii) of the enzymecomposition is derived from a member of the genus Aspergillus such ase.g. Aspergillus aculeatus such as e.g. the beta-glucanase encoded bythe sequence encoded by SEQ ID NO: 4 or homologs thereof (e.g., abeta-glucanase having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4).
 14. The process of any of claims 7 to 13, wherein the pectate lyase(iv) of the enzyme composition forms part of a multicomponent enzymecomposition comprising pectate lyase, xylanase and cellulase activitiessuch as e.g. Novozym 81243™.
 15. The process of any of claims 7 to 14,wherein the mannanase (v) of the enzyme composition is anendo-mannosidase derived from the genus Bacillus such as e.g. Bacillusbogoriensis such as e.g. the endo-mannosidase encoded by SEQ ID NO: 6 orhomologs thereof (e.g., an endo-mannosidase having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to SEQ ID NO: 6).
 16. The process of any ofclaims 7 to 15, wherein the amylase (vi) of the enzyme composition is analpha-amylase derived from the genus Rhozimucor such as e.g. Rhizomucorpusillus such as e.g. the alpha-amylase encoded by SEQ ID NO: 5 orhomologs thereof (e.g., an alpha-amylase having at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5).
 17. The process of any of claims 1to 16, wherein the protease of the enzyme composition is present at aratio between 0-20% w/w, such as e.g. about 5-15%, or about 10% w/w ofthe total enzyme protein.
 18. The process of any of claims 1 to 17,wherein the lipase of the enzyme composition is present at a ratiobetween 0-10% w/w, such as e.g. about 2.5-7.5%, or about 5% w/w of thetotal enzyme protein.
 19. The process of any of claims 1 to 18, whereinthe beta-glucanase of the enzyme composition is present at a ratiobetween 0-30% w/w, such as e.g. 10-20%, or about 15% w/w of the totalenzyme protein.
 20. The process of any of claims 7 to 19, wherein thepectate-lyase of the enzyme composition is present at a ratio between0-10% w/w, such as e.g. 2.5-7.5%, or about 5% w/w of the total enzymeprotein.
 21. The process of any of claims 7 to 20, wherein the mannanaseor amylase of the enzyme composition is present at a ratio between 0-10%w/w, such as e.g. about 2.5-7.5%, or about 5% w/w of the total enzymeprotein.
 22. The process of any of claims 1 to 21, wherein thecellulolytic background composition is present in the enzyme compositionat a ratio between 40%-99% w/w, such as e.g. between 50%-90% w/w, suchas e.g. 60%-80% w/w, such as e.g. 65-75% of the total enzyme protein.23. The process of any of claims 1 to 22, wherein the enzyme compositionfurther comprises one or more enzymes selected from a cellulase, an AA9polypeptide, a hemicellulase, a cellulose inducible protein (CIP) anesterase, an expansin, a ligninolytic enzyme, an oxidoreductase, apectinase, a protease, and a swollenin.
 24. The process of claim 23,wherein the hemicellulase is one or more enzymes selected from axylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.